1. Field of the Invention
The present invention relates to immunoassay techniques for the direct determination of free analytes in biological fluids, and in particular, to immunoassay techniques to effect direct determination of a free analyte which overcome biases caused by excess bound analyte and unbound natural receptors present in the sample.
2. Description of the Prior Art
For the purpose of this invention, a free analyte is that portion of unbound analyte in a biological sample wherein a binding equilibrium exists between the analyte and one or more specific binding receptors naturally occurring in the sample. Ordinarily, the analyte will be firmly and reversibly bound to its receptors and the portion of bound analyte will significantly exceed the portion of free analyte in the binding equilibrium.
Numerous physiologically active and inactive substances that circulate in biological fluids such as blood or serum are firmly and reversibly bound to specific protein carriers or other receptors naturally occurring in the fluid. These substances may belong, for example, to the groups of hormones, steroids, drugs, drug metabolites, proteins, polypeptides, vitamins, toxins and alkaloids. A binding equilibrium state exists in the fluid between the substance and its receptors leading to the distribution of the substance into a bound portion and an unbound or free portion.
For most physiologically active substances, it is known that the free portion is involved in controlling the physiological response associated with those substances. During metabolic activity, which involves the consumption of a free circulating substance by tissues, the concentration of that free substance is maintained constant by the equilibrium mechanism. Therefore, determination of free analyte in biological samples is often clinically more relevant than determination of total analyte, which includes both the free and the bound portions.
A specific example may be given from the field of thyroid disease diagnosis. The thyroid hormones, thyroxine (T4) and tri-iodothyronine (T3), circulate in blood while bound to three major binding proteins: thyroxine binding globulin (TBG), thyroxine binding prealbumin (TBPA) and albumin (Alb). The bound portion of either T4 or T3 is greater than 99.5% of the total hormone, and normally, the free portion is smaller than 0.05% or 0.3%, respectively. It is evident in this case that determination of the free hormone would provide completely different information than the determination of the total hormone.
It is well known that the nature, and often the severity, of thyroid disease status is better correlated with the free thyroid hormone concentration than with the total or protein-bound thyroid hormone concentration. Furthermore, the evaluation of thyroid status in certain other conditions is more clinically efficient using the free thyroid hormone levels. Examples for such conditions are pregnancy or estrogen therapy which may lead to altered levels of total thyroid hormone and/or binding proteins.
Competitive analyte binding methods, particularly competitive binding immunoassays, are used to determine the total concentration of analytes in biological samples such as blood, plasma or serum. These methods apply immunoreactive species such as antibodies to the analyte and cross-reactive analyte conjugates to enable the measurement. In certain cases, special optimizations or the addition of releasing agents are required in order to effect the complete release of the analyte from the endogenous receptors in the sample. In comparison, immunoassay methods for determination of free analytes are designed to avoid biases frequently caused by the interaction of excess bound analyte and unbound endogenous receptors with the added immunoreactive species of the immunoassay.
Numerous patents have been issued that disclose assays for free species of substances such as thyroxine and tri-iodothyronine that are present in clinical samples in both the free and bound forms.
U.S. Pat. No. 4,366,143 to Midgley, et al. discloses a one-step immunoassay method for determination of free analyte concentration, for example, thyroxine (T4). The sample is combined simultaneously with known amounts of a labeled derivative of the ligand or analyte and a specific receptor such as an antibody. After an incubation period, the receptor is separated, and the concentration of free analyte is related to the amount of labeled analyte which becomes associated with the receptor.
The labeled derivative of the analyte is required to be bindable by the added specific receptor and to be substantially non-reactive with the naturally occurring receptors in the sample. This labeled derivative is chemically modified, for example, by providing a blocking bridge between the analyte and the label moieties so as to render it substantially non-reactive with the endogenous receptors, while retaining its ability to bind to the added specific receptor.
U.S. Pat. No. 4,292,296 to Parsons discloses a two-step immunoassay method for determination of free analyte concentration. In the first step, the sample is contacted with a specific receptor for the analyte, for example, an antibody coated on the interior of a test tube. The antibody is chosen to have high affinity constant and low binding capacity in the assay. After a first incubation period, the sample-containing liquid is removed and discarded, and the test tube is washed. In the second step, a labeled derivative of the analyte or a labeled analog of the analyte is added to the washed coated tube to bind with the remaining immobilized receptor in the test tube. After a second incubations period, the liquid phase is separated and the concentration of free analyte is related to the amount of labeled derivative associated either with the coated tube or the removed liquid.
U.S. Pat. No. 4,391,795 to Pearlman discloses a two-step immunoassay method for determination of free thyroxine in a serum sample. In the first step, the serum sample is contacted with an immobilized receptor, preferably anti-thyroxine anti-body coated on a test tube, to bind the free thyroxine hormone. After first incubation period, the sample is removed and a labeled thyroxine hormone, preferably radiolabeled, is added to bind with the remaining unbound receptor. The labeled thyroxine hormone is added in excess of the remaining binding sites of the coated binder. After a second incubation period, the liquid phase is separated, and the concentration of free thyroxine hormone in the sample is related to the amount of labeled thyroxine hormone either present in the coated tube or present in the separated liquid phase.
U.S. Pat. No. 4,410,633 to Hertle, et al. discloses a one-step immunoassay method for determination of free thyroxine or tri-iodothyronine in a liquid sample. This one-step immunoassay method is similar to the Midgley method described above in U.S. Pat. No. 4,366,143. The method uses anti-thyroxine antibody which is immobilized on a solid phase as the receptor. The labeled thyroxine derivative is a thyroxine-linked horseradish peroxidase which does not significantly interact with the thyroxine binding globulin or thyroxine binding prealbumin present in the sample. The enzymatic activity of the horseradish peroxidase label is measured.
In general, hapten-enzyme conjugates, including thyroxine-enzyme conjugates, are well known in the prior art. Specific enzyme immunoassay procedures, which utilize these conjugates, are applied for quantitation of total analyte concentration in biological samples. See, for example, U.S. Pat. Nos. 3,839,153, 3,850,752, 3,879,262 and 4,040,907.
However, in these patents, as well as in the general literature on competitive binding immunoassays, there are no suggestions or disclosures that these procedures are suitable for the determination of free hapten concentration and certainly not free thyroxine.